Origin and Specialization of Splenic Macrophages

A-Gonzalez, Noelia1* and Castrillo, Antonio2,3

1
Institute of Immunology. University of Münster, 48149 Münster, Germany
2
Instituto Investigaciones Biomédicas “Alberto Sols”, IIBM Madrid, Spain
Centro Mixto Consejo Superior de Investigaciones Cientificas y Universidad Autonoma de
Madrid (IIBM CSIC-UAM).
3
Unidad De Biomedicina (Unidad Asociada al CSIC), IIBM- Universidad Las Palmas de Gran
Canaria, ULPGC. Grupo de Investigación en medio ambiente y Salud (GIMAS), Instituto
Universitario de Investigaciones Biomedicas y Sanitarias (IUIBS, ULPGC).

* Corresponding Author: alonsogo@uni-muenster.de

Abstract
Macrophage heterogeneity in the spleen has been long documented, with four subsets
populating the different splenic compartments. The diverse environments on the splenic
compartments determine their varied phenotype and functions. In the white pulp, highly
phagocytic macrophages contribute to the generation of the immune response. The marginal
zone contains two populations of macrophages, which also contribute to the immune
response. Their strategic position in the bloodstream and their unique phenotype confer them
a crucial role in the defense against blood borne pathogens, placing them at the crossroad
between innate and adaptive immune responses. Macrophages in the red pulp are classically
linked to homeostatic and metabolic functions in erythrocyte phagocytosis and iron recycling.
We review here recent advances demonstrating the importance of macrophage ontogeny and
organ development in determining the phenotype, transcriptional profile and, ultimately, the
functions of the populations of splenic macrophages.
1. Introduction
Composed by both connective and lymphoid tissue, the spleen is a complex organ with a
highly organized compartmentalization and an intricate microcirculatory system [1]. The
particular microanatomy of the murine spleen makes it a unique organ which harbors
heterogeneous populations of stromal, endothelial and immune cells. In the red pulp, venous
sinusoids are mixed with leukocytes, stromal cells and splenic cords creating a network that
allows the selection of effete red blood cells and unwanted material by professional
phagocytes [2]. The white pulp is a dense lymphoid tissue compartment in which B cells are
organized in follicles, surrounded by T cell areas, where the immune response and the
generation of antibodies take place within the germinal centers [1]. The marginal zone of the
murine spleen is formed by a sinusoid system of sinus lining cells that surround the white
pulp, where the blood freely percolates on its way to the red pulp. These areas were believed
to be transitional zones, with migrating populations, however, in the mouse spleen, several
unique populations of macrophages, B cells and endothelial cells are permanent residents of
the marginal zone [3]. Hence, to understand the variety of immune cell types in the different
compartments of the spleen, the coexisting microenvironments must be considered. Those
specific environmental cues ensure the multifaceted mechanisms that allow the spleen to
perform different immune and homeostatic functions. Similarly, the concomitant
microenvironmental signals potentially influence the different immune cell populations found
in the various compartments of the murine spleen. In this review we will focus on the variety
of splenic resident macrophages, with a particular emphasis on the murine system.
A simple definition of the term “macrophage” is currently a challenge due to the continuous
advances in the field, with the definition of their multiple origins [4, 5, 6, 7], the study of their
plasticity [8] and reprogramming capacities during disease [9], and with the characterization
of novel subsets [10, 11]. Macrophages are yet considered a myeloid cell type, together with
granulocytes, monocytes and dendritic cells. All myeloid linages were classically defined as
hematopoietic cells that derive from a single clonogenic common myeloid progenitor [12].
This seminal report described a common myeloid progenitors that gave rise to exclusive
megakaryocyte/erythrocyte or granulocyte/macrophage progenitors and subsequently
differentiated into erythrocytes, granulocytes, monocytes, dendritic cells (DCs) and
macrophages. While for some time these progenitors were believed to give rise to all tissue
myeloid cells subsets, it is now accepted that the generation of some tissue resident
macrophages occurs before the establishment of definitive hematopoiesis and develop
independently of the common myeloid progenitors of the bone marrow [4, 5, 6, 7]. As
mentioned above, in the last decade, a prolific literature has been released about the origin and
establishment of tissue resident macrophages, implicating multiple factors, such as
transcriptional regulators, microenvironment and plasticity (recently reviewed in [13, 14, 15]).
Many efforts have been done to characterize the myeloid cell subsets in the spleen, both in the
steady state and during disease. Due to the intrinsic antigen presentation capabilities and
migratory behavior of many myeloid cells, a plethora of these subsets can be identified in the
spleen under different circumstances, substantially complicating the scenario [16]. In addition,
the aforementioned coexisting microenvironments in the spleen create the perfect niches for
the homing of heterogeneous myeloid populations into the different compartments. In fact,
four different populations of resident macrophages are found in the three compartments of the
murine spleen [17]. Hence, it perfectly resembles the influence of the microenvironment on
tissue-resident macrophage heterogeneity in a single organ, through diverse macrophage
adaptations to each compartment. We will review here the ontogeny, phenotypic profile and
functional specialization of the different macrophage subsets of the murine spleen with a
special focus on the influence of the microenvironment in their transcriptional activity.

2. Cellular and molecular mechanisms that regulate splenic development

As a secondary lymphoid organ, certain aspects during the splenic embryonic and early
postnatal development are common to lymph node organogenesis. The initiation of lymphoid
organogenesis requires the spatiotemporal coordinated action of cytokines, chemokines, and
transcription and angiogenic factors [18]. The interplay of cytokines (importantly
lymphotoxin-α (LTα)), chemokines (e.g. CXCL13), transcription factors (e.g. RORγ) occurs
mainly within two cell types: a subtype of innate lymphoid cell, known as lymphoid tissue
initiating cell (LTi), and the stromal lymphoid tissue organizer cells (LTo) [19, 20]. On the
first stages of lymphoid organogenesis, retinoic acid (RA) is produced by nerve fibers and
induces the expression of CXCL13 in mesenchymal cells that, in turn, attract LTi cells to
form the organ anlage [21]. These cells produce high amounts of LTα and β, which activate
LTβR signaling pathways in LTo cells, promoting the expression of cytokines, chemokines
and adhesion molecules crucial for the fully organization and maturation of the lymph nodes
[22]. These events, together with the exposure to the external environment after birth,
complete the maturation of the lymphoid organs. Although the previously mentioned
sequence of events applies to the majority of secondary lymphoid organs, some differences on
the molecules involved, specific signaling pathways and temporal events have been described
for Peyer’s Patches (PPs) and spleen development. For example, a recent study shows that
lymph node, but not PPs or spleen, organogenesis is dependent on LTi-cell-mediated
activation of lymphatic endothelial cells, instead of mesenchymal LTo, which is a subsequent
event [23]. Nonetheless, as mentioned above, the spleen is a particular case among secondary
lymphoid organs, due to their multiple functions, including their ability to act as an
hematopoietic organ during development, and even in special occasions in the adult life.
These factors influence its organogenetic process and certainly impact the establishment of
the myeloid and lymphoid populations. Particularly, the definitive compartmentalization and
establishment of the splenic resident macrophages are complete between the 3rd to 4th week of
the postnatal development [24].
The spleen develops from the mesoderm, starting at embryonic day E12 in mice, with the
appearance of the splanchnic mesodermal plate, considered as the splenic anlage[1]. Several
transcription factors have been shown to be crucial for these first steps on splenic
development, such as Pbx1, Hox11 (Tlx1), Nkx3.2 (Bapx1) and Pod1 (capsulin, Tcf21) [25].
Particularly, Tlx1 is crucial to regulate RA signaling in the developing spleen [26]. Around
day E13.5-E14 LTi cells can be found in the spleen, suggesting similarities with the initial
developmental steps of the lymph nodes. However, these cells do not express LTα in the
neonatal spleen and only B cells at 4 days after birth do, underlying different pre- and
postnatal developmental pathways in the spleen [27]. Although in an LTαβ independent
manner, splenic neonatal LTi are believed to contribute to the white and red pulp segregation
through their interaction with MAdCAM-1+CD31+CD201+ spleen stromal organizer cells
[28]. In contrast, in the adult spleen, LTαβ producing LTi cells were reported to be crucial for
the maintenance or B and T cell compartmentalization through their interaction with LTβR
expressing endothelial cells [29]. Nevertheless, during cytomegalovirus (CMV) and
lymphocytic choriomeningitis virus infections, both of which cause disruption of the B and T
cell compartmentalization, the maintained expression of lymphoid chemokines needed for B
and T cell areas recovery occurred through LT-independent pathways, suggesting a
reactivation of the embryonic program under stress conditions [30, 31, 32].
Hence, the different developmental and organizational pathways during embryonic and
postnatal phases must be considered so as to understand the establishment of the different cell
populations in the spleen. Remarkably, different macrophage subsets populate the murine
spleen at different stages during embryonic and postnatal development, following the
compartmentalization and structural events of the different areas. . Indeed, macrophages that
resemble the red pulp macrophage population by their expression of F4/80 are present since
embryonic stages but only locate in the red pulp between postnatal day 14-21, when the
definitive red and white pulp segregation is patent; marginal zone macrophages (MZMs) and
marginal metallophilic macrophages (MMMs) appear only after birth, around postnatal day 7-
14, and are located at their areas between days 21-28 after birth [24]; and tingible body
macrophages (TBMs) are only visible in the white pulp once the germinal centers are active
through their contact with the external environment after weaning (Fig. 1). Therefore, the
ontogeny and development of the different resident macrophage populations of the spleen is
also influenced by the various mechanisms of splenic development, ultimately influenced by
the microenvironment. The few animal models available with myeloid-specific deficiencies in
molecules implicated in spleen development reveal mild alterations on spleen development.
However, the expression of these ontogenic factors in macrophages seems to be important for
the maintenance of the splenic adult structure. Remarkably, a work from Mountz and
collaborators in 2015 addressed the contribution of myeloid cell derived LTβR to macrophage
establishment in the marginal zone. The study shows that macrophages in the marginal zone
of the spleen were fully developed one month after birth but suffer slight disruption as they
age. Yet, a correct compartmentalization of the spleen was observed in these mice. The
authors also described an increased germinal center reaction in aged Ltbrfl/flLys-Cre, probably
due to the accumulation of apoptotic cell-derived antigens in the marginal zone as a
consequence of MZM disruption over time [33]. Altogether, these data suggest that, although
the correct establishment of macrophages in the spleen occurs in parallel with organ
development, macrophage-derived factors seem to be dispensable for the correct splenic
organogenesis. In addition, analysis of the recombination efficiency of several Cre- myeloid
drivers, using several mouse strains, showed a low recombination efficiency of the Lys-Cre
promoter, in spleen macrophages, indicating that the Loxp/Lys-Cre system might not be ideal,
at least under steady-state conditions, to study the functions of splenic macrophages [34].

3. Functional specialization of macrophages in the spleen

As previously exposed, immense advancements in the molecular definition of tissue-resident
macrophages, based on their ontogeny and microenvironment, have been done. Yet, the
homeostatic and immunological significance of all these concepts in selected macrophage
populations in specific niches need further investigation. One of the current challenges is to
define whether specific macrophage functions, are dictated by their ontogeny, at specific
locations and whether tissue-resident macrophages can be reprogrammed by novel
environments in the adult. The current scenario requires further investigations; yet, some
evidences already underlie a crosstalk between macrophages and tissue microenvironment in
the adult that shapes macrophage functions and contribute to organ integrity [35]. Particularly
intriguing is the case of the spleen, with the multiple scenarios and macrophage subsets that it
harbors. Here we give an overview of the trophic and immunological functions of the splenic
macrophage populations connected to their ontogeny and tissue microenvironmental cues.

3.1. “Metabolic”: Red Pulp Macrophages

Red pulp macrophages most known function is the clearance of effete red blood cells that are
trapped within the red pulp sinusoids and stromal network. These cells reside exclusively in
the red pulp and are identified as F4/80+VCAM1+CD11blo by flow cytometry. The molecular
mechanisms by which senescent erythrocytes are recognized by RPMs are not completely
understood. They are, nonetheless, equipped with the adequate machinery to process and
metabolize heme and Iron after senescent erythrocyte clearance. High levels of expression of
the scavenger receptor CD163, Spi-C, VCAM1, Heme Oxigenase (HO-1) and ferroportin, by
RPMs ensure their regulation of iron metabolism [36].
The metabolic function of RPMs is clearly influenced by the environment and linked to the
transcriptional regulation of their developmental pathways. RPMs develop in mice during
embryonic and fetal stages, with very few, if any contribution of monocytes. However, after
macrophage depletion, bone marrow monocytes are able to repopulate the red pulp with
F4/80+VCAM1+ macrophages [37]. RPMs were shown to derive partially from yolk sac
macrophages [38], with the contribution of fetal definitive hematopoiesis [39]. However, the
precise fetal progenitor from which RPM derive is not completely determined.
Heme regulates the expression of Spi-C, a transcription factor responsible for the
development of RPMs. Spi-C, subsequently regulates the expression of VCAM1, influencing
as well in iron metabolism. However, the expression of HO-1 and ferroportin seem to be
directly influenced by Heme and independent of Spi-C [40]. Ultimately, the crosstalk between
erythrophagocytosis, iron recycling and RPM transcriptional activation is needed to the
maintenance of these populations in the adult spleen. Numerous evidences point to the
existence of a different mechanism of damaged erythrocyte elimination, through a process
called eryptosis that involves exposure of phosphatidylserine in the outer membrane and
shrinking of the cell, in an analogous fashion that nucleated cells suffer apoptosis [41]. These
eryptotic erythrocytes are, however, believed to be recognized and eliminated by different
subsets of phagocytes in the spleen [42].
RPMs have also been shown to perform immunological functions, in response to
inflammatory stimuli and parasite infections. Using Spic-/-, Kim and colleagues showed that
RPMs produce high amounts of type I interferons, playing a key role in the defense against
Plasmodium chabaudi [43]. In vitro studies using isolated RPM in co-cultures with CD4+ T
cells have revealed an implication of RPMs in the process of differentiation or T cells towards
regulatory T cells [44].

3.2. “Phagocytic”: White Pulp Macrophages

The white pulp has for many years been considered as the immunological compartment of the
spleen. Now we know that the red pulp also perform some immunological functions and the
marginal zone is as well a dynamic area where specific immune responses take place. Within
the germinal centers, F480negCD68+ macrophages containing phagocytosed apoptotic cells
are frequently found. Condensed chromatin fragments are visible inside these macrophages
displaying a dark blue staining pattern when using hematoxylin and eosin. Consequently,
these macrophages were originally called tingible body macrophages (tingible meaning
stainable). TBMs are analogous to macrophages found in the germinal centers of lymph nodes
and PPs, as well as in the thymus of adult mice, during its physiological involution. In the
spleen, their main function is the phagocytosis of apoptotic B cells generated in the germinal
centers during antigenic responses, where hyper proliferation and somatic hypermutation of
these cells take place [45]. In addition, germinal centers are also the sites of B cell self-
renewal and clonal selection, crucial to maintain tissue homeostasis. During the high affinity
maturation of the clonal selection process, autoreactive B cells are also eliminated by
apoptosis and cleared by TBMs. Hence, the highly phagocytic macrophages in the white pulp
play a dual role as homeostatic regulators and actively participating of the immune responses
against antigens.
Certainly, the white pulp environment shapes the phagocytic aspect of these macrophages.
Several soluble molecules required by the macrophage to engage the apoptotic cell during its
recognition phase are highly expressed in the splenic white pulp, such as Mfge8, Gas6, or
C1q. TBMs as well express high levels of phagocytic receptors as compared with the other
macrophage populations in the spleen, for example, CD68, Mertk, Timd4 or CD36 [17]. All
these molecules have been previously shown to play decisive roles in the phagocytosis of
apoptotic cells, however, whether their elevated expression in this macrophage subset is
influenced by specific white pulp derived signals is still unclear. Nonetheless, we have
previously shown that the constant feeding of macrophages with apoptotic cells promote the
expression of Mertk, Gas6 and CD36 [46]. Hence, one possible scenario is that the constant
phagocytosis of B cells in the germinal centers provides the signals to shape the phenotype
and function of TBMs, contributing to maintain peripheral B tolerance and regulating the
germinal center reaction, ultimately, ensuring the correct defense against antigen-driven
inflammatory processes.

3.3. “Immune”: Marginal Zone Macrophages

The marginal zone of the murine spleen is a dynamic structure located between the red and
white pulps where the blood is shed before percolating to the red pulp. Its specific location
and cell composition create an adequate environment for the recognition and clearance of
blood borne antigens. In addition, it is the starting point in the generation of the immune
response against T cell independent antigens. The marginal zone contains a special stromal
endothelial cell, called the marginal sinus lining cell that expresses MadCAM1. In contrast to
MadCAM1+ endothelial cells in the lymph nodes, splenic marginal sinus lining cells do not
form blood or lymphatic vessels, but they line up forming an outer ring around the white
pulps. Closely associated to these endothelial cells, concentrate the CD169+ MMMs and
outer, several layers of MZMs coexist with specialized B cells and dendritic cells, supported
by a network of stromal conduits. Remarkably, the marginal zone is the only microanatomical
environment in which two phenotypically and functionally different macrophage populations
coexist. Both populations of macrophages are responsible for the clearance of blood-borne
encapsulated bacteria, such as meningococci [47], Campylobacter jejuni [48] or Streptococus
pneumoniae [49]. They have also been shown to phagocytose parasites, like Leishmania
donovani [50]. Copious speculations about their separate functions were made in the past, still
being a controversial topic. Conditional depletion of MMM or MZM via diphtheria toxin
receptor, using Cd169DTR and Cd11cDTR transgenic mice, showed no conclusive results on
their selectivity of splenic macrophage depletion [51]. Another caveat in the study of these
populations is the difficulty to isolate them and the lack of mouse models deficient
specifically on one of them. The isolation of macrophages of the marginal zone still remains a
challenge due to their small frequency in the spleen, their close contact with endothelial and
stromal cells and the particular properties of their surface markers. MMMs and MZMs are
defined by the high expression of the lectins CD169, Sialoadhesin, and SIGNR1 respectively.
These lectins get rapidly internalized by the macrophage during sample preparation for flow
cytometry or sorting techniques, which hinders the application of high throughput techniques
to define their transcriptomic profile. Nevertheless, different functions have been proposed for
these two macrophage populations:

3.3.1. Marginal Metallophilic macrophages

Their high expression of CD169, Sialoadhesin, similar to the subcapsular sinus macrophages
in the lymph nodes suggest analogous functions, however the different disposition of both
populations in the organs and the particular anatomy and environmental cues of the spleen
confers them unique features.
MMMs have been selectively implicated in the degradation and clearance of viruses, such as
porcine reproductive and respiratory syndrome virus [52]. An elegant study from den Haan
and colleagues showed that CD169+ MMMs specifically induced T cell responses. Using
conjugated OVA-antibodies to target different antigen presenting cells in the spleen, the
authors demonstrated that MMM can specifically take up antigens, transfer them to CD8+
dendritic cells, ultimately activating T cells [53], showing their potential as antigen presenting
cells and their direct implication in the generation of the adaptive immune response.
Besides their immunological functions on the recognition of blood-borne pathogens and
antigens, processes dictated by their surrounding microenvironment, MMMs also perform
trophic functions by actively interacting with their neighboring cells. Follicular B cells are
essential to the maintenance of MMMs through the release of LT [54]. In addition,
follicular B cells have been shown to migrate towards the marginal zone, in response to
oxysterols, suggesting the idea that MMMs are producing those oxysterols [55]. Together
with the fact that LXR, a transcription factor activated by oxysterols, drives the
differentiation of MMMs, this would potentially contribute to understand how metabolic
pathways actively influence the differentiation of macrophages in the marginal zone [24].
The close association of MMMs with the MadCAM1+ endothelial sinus lining cells suggests
a cooperative role for these macrophages in the organizing functions of the sinus lining cells
in the maintenance of the splenic structure. This was in principle supported by evidences of
mouse models lacking MMM, which also showed disrupted splenic compartmentalization,
with disorganized B cell follicles [56]. However, we showed in LXR-/- mice, where there is
total absence of cd169+ MMM, no apparent affection of sinus lining cells and a proper
organization of the B and T cell areas [24], ruling out an organizational function for these
macrophages.
3.3.2. Marginal zone macrophages

The immunological role of MZMs is highly determined by the nature of the antigens that they
phagocyte from the blood stream and their close interaction with MZ-B cells. These
specialized B cells recognize blood-borne antigens and migrate to the follicles in the white
pulps to present them to FDCs, which ultimately generate the germinal center reaction. SIGN-
R1 expressed by the MZMs has revealed as a key molecule in the interaction between MZ-B
cells and MZMs. In the absence of MZ-B cells there is a downregulation of SIGN-R1 in
MZMs [57] and, reciprocally, when SIGN-R1 is not present, the migration of MZ-B cells to
the follicles is impaired, therefore, altering their interaction with FDCs and germinal center
reaction [24, 58]. Moreover, the scavenger receptor MARCO, which is also highly expressed
by MZMs, had been previously shown to interact with B cells promoting their maintenance
and correct positioning in the marginal zone [59]. This crosstalk between the MZMs and MZ-
B cells is crucial to ensure the correct immune responses against T cell independent antigens,
contributing to maintain central tolerance. Furthermore, it is crucial to understand MZM
intrinsic plasticity in response to environmental cues. Using the LXR-/- model, we observed
that in the absence of MZMs clearance of blood borne pathogens was taken over by RPMs,
underlying the plasticity of those macrophages. However, these mice exhibited a blunted
immune response, with a decreased generation of specific antibodies IgM antibodies, against
T cell independent antigens, suggesting that physical communication with MZ-B cells is still
needed to generate the immune response. These results suggest that, although splenic
macrophages possess the ability of reprogramming some of their functions, the interplay with
the environment is crucial to determine the specific functions of the different macrophage
subsets.
MZMs are also implicated in central tolerance through phagocytosis of blood-borne apoptotic
cells. Several studies showed that intravenously injected apoptotic cells were rapidly
phagocytosed by MZMs [60, 61]. This prompted the suppression of an immune response
against dying cell associated antigens, leading to the maintenance of immune tolerance.
Interestingly, the interaction of LTαβ expressed by MZ B cells and LTβR expressed by
MZMs, was shown to be important for efficient engulfing of circulating apoptotic cells by
MZMS in lupus disease [33]. Remarkably, these studies underlie the importance of MZMs in
the control against developing autoimmune diseases, in a similar fashion as of TBMs. In both
cases, the molecules implicated in this process are not fully identified; however the different
origin of the cells that they phagocytose may lead to different transcriptional program on each
macrophage population, raising the heterogeneous properties of splenic macrophages.
Supporting this theory, in a recent report, we showed that different environments contribute to
increase tissue-resident macrophage heterogeneity through the phagocytosis of unwanted
circulating cells [11].

4. Transcriptional landscape of splenic macrophages origin

Tissue resident macrophages show diverse origins and differentiation pathways influenced by
the environment, which impacts their transcriptional profile and activation landscapes [13,
39]. The integration of single-cell RNA and ATAC (assay for transposase-accessible
chromatin) sequencing (RNA-seq and ATAC-seq), cytometry by time-of-flight mass
spectrometry (CyTOF), genetic fate mapping, and functional analyses using cutting edge
techniques, such as CRISPR-Cas9, enormously contributes to a deep phenotypic
characterization of the diverse tissue resident macrophage populations. This is now basic to
understand their differentiation and activation, also during disease [15]. Over the last few
years, huge advances have been made to define the transcriptional programs of macrophages
during development, which has contributed as well to understand the influence of the
microenvironment in their reprogramming capacities [8]. Several transcription factors have
been described as part of a common macrophage core signature during developmental phases.
Multipotent erythro-myeloid progenitors (EMPs) expressing the transcription factor c-Myb are
found in the embryo at day E8.5 and were shown to give rise adult tissue resident
macrophages through primitive and definitive hematopoiesis [62]. To define the complete
cascade of transcriptional events in macrophages, several studies explored the genome-wide
locations of enhancers and master regulators of myeloid precursors and macrophages. PU.1 is
considered as a master regulator of macrophages occupying most enhancers [63]. Maf and
Mafb transcription factors are also implicated in the regulation of macrophage differentiation
and proliferation [8]. In addition to these transcriptional master regulators, different
macrophage populations are specifically dependent on transcription factors for their
development and differentiation. For example, the transcription factor Gata6 is essential for
the differentiation of peritoneal macrophages [64] and the nuclear receptor PPARγ is crucial
for alveolar macrophage identity [65]. Particularly, in the spleen, Spic and IRF8 regulate the
development of red pulp macrophages [66-68] and LXRα of marginal zone and metallophilic
macrophages [24].
Whether organogenesis influences macrophage development or vice versa on each particular
organ is still an open debate in the field. A report in Science in 2016 suggests that macrophage
differentiation is an integral part of organogenesis, based on the description of a novel
macrophage precursor, pre-macrophages or pMacs, that colonizes the whole embryo at E9.5
and ultimately specify into tissue-resident macrophages in the different organs that they study
[69]. Yet enlightening and intriguing, this study does not include adult splenic macrophages in
the screening; hence still the question of whether the four splenic macrophage populations
share a common precursor and primitive transcriptional profile remains unknown. As
mentioned above, several transcription factors have been described to regulate the
development of three splenic macrophage subsets:

4.1. Spi-C and IRF8: red pulp macrophages

Spi-C was the first transcription factor identified that controls the development of RPMs.
Murphy and colleagues showed a dramatic reduction of RPMs in Spic-/- mice with almost no
affection of other macrophage populations in the spleen [65]. The authors showed that Spi-C
played a cell-autonomous effect in the development of RPMs, since it was corrected by
retroviral Spi-C expression in reconstitution studies through bone marrow transplant. Spi-C
expression is controlled by heme through the degradation of its transcriptional repressor
BACH1 [70]. Spic-/- mice accumulate large amounts of heme and iron in their splenic red
pulp, therefore showing splenomegaly and defects in iron metabolism. Interestingly, the same
group later showed that Spic was also implicated in the development and maintenance of bone
marrow F480+VCAM1+ macrophages, a subtype of bone marrow resident cells with iron-
recycling capacities similar to RPMs, also influenced via heme and BACH1 regulation [70].
Remarkably, these studies demonstrate a direct link between Spi-C-dependent transcriptional
activity and iron metabolism in the development of splenic and bone marrow macrophages.
There are, nonetheless, several interesting unexplored paths regarding Spi-C activity. For
example, the direct Spi-C downstream targets that drive the differentiation of monocytes into
RPMs are still not well-defined. Studies that explore the genome-wide Spi-C binding
locations, along with RNA-seq experiments would possibly shed light on these questions.
The role of other transcription factors, such as IRF4 and IRF8, implicated in the development
of multiple hematopoietic cells (for example CD11b+ dendritic cells [71] and B cells [68, 72]
respectively) has been reported to be important for the development of RPMs. Indeed, the
lack of both factors causes a dramatic decrease of the RPM population [68].
4.2. LXR: macrophages in the marginal zone
Despite the technical hitches in the isolation of marginal zone macrophages explained in point
3.3., the combination of imaging techniques and in vivo functional analysis with the use of
diverse mouse models, allow us and others to define crucial molecules for their development
and establishment. In the late nineties, with the generation of different knock-out and
transgenic mouse models of the lymphotoxin (LT)/tumor necrosis factor (TNF) family, the
implication of these cytokines on the development of marginal zone macrophage populations
was evidenced (reviewed in [56]). However, the mouse models lacking both populations of
marginal zone macrophages, LTα-/-, LTβ-/-, and LTβR-/-, showed also profound defects in
secondary lymphoid organ development, with absent lymph nodes and PPs, and disorganized
B and T cell zones in the spleen [73, 74]. Hence, the specific role of these cytokines in
macrophage development is not completely clear, and macrophage deficiencies are believed
to be consequence of the organogenetic impairment observed in those animals.
To date, the only transcription factor described to be responsible for the development of
macrophages in the marginal zone is the transcription factor LXRα, a member of the nuclear
receptor superfamily. We showed that Lxrα-/- mice selectively lack MMMs and MZMs but
showed RPMs and TBMs in their appropriate splenic locations. Remarkably, lymph node
macrophages, defined by the expression of the same surface lectins, were not affected in these
animals. In an effort to identify the specific progenitor of these macrophages, we performed
different functional analysis using bone marrow chimeras, characterization of bone marrow
progenitors and transferring isolated monocytes. We showed that adoptively transferred
LXRα-sufficient CX3CR1intLy6Chi monocytes were able to populate the marginal zones of
Lxrα-/- mice and to differentiate into both macrophage subsets [24]. We concluded that, at
least when the marginal zone space lacks macrophage populations in LXRα-deficient mice,
MMMs and MZMs can develop correctly from bone marrow monocyte precursors, pointing
out to definitive hematopoiesis as responsible for the ontogeny of MZ- and MM-
macrophages. Fully differentiated MMMs and MZMs start to appear in the postnatal murine
spleen, and are only properly located in the marginal zone after weaning, likely ruling out
primitive hematopoiesis as their primary source. However, to definitely exclude primitive
macrophages as precursors of MZ macrophages in adult mice under homeostasis, additional
studies using parabiosis experiments in WT and LXR-deficient mice will be required.
Whether macrophages in the marginal zone might have a unique monocytic origin and
whether the specific transcriptional pathway by which LXRα might be directly responsible for
it still remains to be clarified. One possibility is that oxysterols, the endogenous ligands of
LXR, in the spleen activate LXRα to drive the differentiation of monocytes to marginal zone
macrophages. However, further studies should be done to clarify these hypotheses.
Interestingly, several studies speculate that macrophages in the marginal zone might be
producing oxysterols that also participate in the regulation of follicular B cell migration [55,
75].

5. Conclusions and future perspectives

The potential communication between the different populations of splenic macrophages has
been proposed to be crucial for their development and functions. For example, the mouse
models with marginal zone macrophage deficiencies normally lack both MMMs and MZMs,
suggesting a strong interdependence between both populations in their developmental
pathways. However, little is known about the potential communication between white and red
pulp macrophages or whether there are common pathways during their development. The
temporal differences in the establishment of the four populations in the spleen suggest
different ontogeny and differentiation pathways. Whether there is a unique progenitor for all
splenic macrophages remains to be clarified. Applying single-cell sequencing to myeloid
progenitors at different stages together with the adult macrophage populations will be useful
to stablish common pathways and transcription profiles, and it would contribute to build a
“molecular map” of the development and differentiation of each population. However, the
different splenic microenvironmental cues that influence the phenotypic landscape of the
macrophage populations in the spleen, have only been partially described. Namely, the
specific environmental cues crucial for marginal zone macrophage development still remain
unknown. During spleen development, stromal cells act as central regulators, although it is
still unknown which specific cues are involved in this process. Interestingly, a recent study
shows that oxysterols produced by stromal fibroblastic cells promoted lymphoid tissue
formation, by signaling through their receptor GPR183 expressed by LTi cells [76]. This
finding underlies the importance of the communication of the microenvironment with
precursors, to establish definitive organogenesis. Similarly, these molecules produced by
stromal cells could directly influence macrophage precursors during their development and
establishment. Whether oxysterols in the marginal zone specifically activates LXR in
monocytes conditioning the establishment of macrophage populations in that area is a
possible mechanism that remains to be explored.
6. Figure Legends

Figure 1. Ontogeny and Establishment of Splenic-Resident Macrophages. Macrophages

in the red pulp originate from primitive yolk sac and fetal definitive hematopoiesis, the latest
through fetal monocytes. In the postnatal spleen, red pulp macrophages are found since early
stages dispersed throughout the yet non-compartmentalized neonatal spleen. Between
postnatal day 7 and 14, primitive lymphoid follicles form and first marginal metallophilic
macrophages appear in a disorganized manner. During the third week of the postnatal spleen
development, white and red pulp compartments start to define, with the concomitant onset of
the marginal zone macrophage populations. At the same time, red pulp macrophages are
excluded from the white pulp areas and the first tangible body macrophages appear coinciding
with the generation of the germinal center reaction in some follicles. At day 28 of the
postnatal development, the spleen shows its definitive microanatomic structure with define
white and red pulps and marginal zone. At this time, the four populations of macrophages
inhabit their compartments. The scheme also shows the transcription factors implicated in the
proper differentiation and establishment of red pulp macrophages (Spi-C and IRF8), their
precursors (PU.1 and Myb) and marginal metallophilic and marginal zone macrophages
(LXRα). E: embryonic day; P: postnatal day; YS: yolk sac; FL: fetal liver; TB: tingible body;
RP: red pulp; MZ: marginal zone; MM: marginal metallophilic; Mo: monocyte; MΦ:
macrophage.
Acknowledgments

Research in Antonio Castrillo´s lab is supported by grants from Spanish Ministerio de Ciencia
y Tecnología [SAF2008-00057], Ministerio de Economia y Cometitividad [SAF2011-29244,
SAF2014-56819-R, SAF2015-71878-REDT] and Comunidad de Madrid I+D [Grant
S2010/BMD-2350 Raphyme-CM and B2017/BMD-3680 Chronobite-CM].
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 HIGHLIGHTS

Origin and Specialization of Splenic Macrophages

Noelia A-Gonzalez and Antonio Castrillo

 The Spleen is the largest secondary lymphoid organ in the body and the largest filter of the
blood.
 The Spleen is crucial for the generation of immune responses to blood-borne antigens and for
the clearance of circulating unwanted material.
 At least four different macrophage subsets populate the distinct microanatomical splenic
compartments under steady-state conditions.
 Macrophage position and function in their particular splenic microdomains confer them
unique phenotypes that are crucial for the defense against blood borne pathogens, clearance of
apoptotic cells and iron metabolism.
 Discrete number of transcription factors, including Irf8, Spic and Nr1h3 control the
differentiation of red pulp macrophages and marginal zone macrophages.